Drug addiction is reported to have adverse effects in male reproduction. Dextromethorphan (DXM) administration was used in this study as a model of addiction in rats, and various treatments including the use of pre-germinated brown rice (PGBR) were investigated for their effects on the changes of sperm quality, testicular structure and androgen receptor (AR) expressions in rats receiving DXM.
Trang 1International Journal of Medical Sciences
2018; 15(9): 921-928 doi: 10.7150/ijms.26076 Research Paper
Recovery effect of pre-germinated brown rice on the changes of sperm quality, testicular structure and
androgen receptor expression in a rat model of drug addiction
Samur Thanoi1,2 , Jureepon Roboon1 and Sutisa Nudmamud-Thanoi1,2
1 Department of Anatomy, Faculty of Medical Science, Naresuan University, Phitsanulok, Thailand
2 Centre of Excellence in Medical Biotechnology, Faculty of Medical Science, Naresuan University, Phitsanulok, Thailand
Corresponding author: Samur Thanoi, PhD., Department of Anatomy, Faculty of Medical Science & Centre of Excellence in Medical Biotechnology, Faculty of Medical Science, Naresuan University, Phitsanulok 65000, Thailand Tel: +66 55 964600; Fax: +66 55 964770; E-mail: samurt@nu.ac.th
© Ivyspring International Publisher This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) license (https://creativecommons.org/licenses/by-nc/4.0/) See http://ivyspring.com/terms for full terms and conditions
Received: 2018.03.15; Accepted: 2018.05.27; Published: 2018.06.12
Abstract
Drug addiction is reported to have adverse effects in male reproduction Dextromethorphan
(DXM) administration was used in this study as a model of addiction in rats, and various treatments
including the use of pre-germinated brown rice (PGBR) were investigated for their effects on the
changes of sperm quality, testicular structure and androgen receptor (AR) expressions in rats
receiving DXM The results demonstrated that these animals showed significant reduction in all
parameters of sperm quality, an increase in abnormal testicular structure and decreased androgen
receptor expression in spermatogenic, Sertoli and Leydig cells However, different effects of the
treatments applied in this study were observed with the greatest recovery effect from treatment
with PGBR Sperm motility and sperm concentration reverted to normal after treatment with PGBR
for 60 days Moreover, all parameters of testicular structure also returned to normal after 60 days
of PGBR treatment, as well as AR expression in Sertoli and Leydig cells Therefore, we have
demonstrated that PGBR treatment can reverse the changes in sperm quality, testicular structure
and AR expression in addicted animals and PGBR may be a novel therapeutic strategy for the
treatment of drug addiction
Key words: Drug addiction, Dextromethorphan, Sperm quality, Testicular structure, Androgen receptor
Introduction
Drug addiction is reported to have adverse
effects on the male reproductive system including
disruption of the reproductive axis [1], reduced
testosterone [2,3] and decreased sperm quality
[4,5,6,7] Furthermore, apoptosis of germ cells [6,7]
and decreased testosterone level [8] can be induced by
methamphetamine
Dextromethorphan (DXM) is anantitussive or
act as adissociativehallucinogen via multiple effects,
including as a nonselective serotonin reuptake
inhib-itor [9] Within the past few years illicit use and drug
abuse of antitussive drugs, especially DXM, have risen Therefore, DXM was used in this study as a model of drug addiction in rat
We previously demonstrated that pre-germ-inated brown rice (PGBR) has a recovery effect on improving sperm quality in a rat model of depression [10] PGBR is a food supplement which contains many effective substances including 𝛾𝛾-oryzanol, α-tocoph-erol (vitamin E), pyridoxine (vitamin B6), thiamine (vitamin B1) and high GABA [10] Therefore, we investigated whether PGBR would be an effective treatment in this study in comparison with other treatments including drug withdrawal, Diazepam (a Ivyspring
International Publisher
Trang 2common drug treatment for addiction), and standard
GABA (gamma-aminobutyric acid) The results from
this study will provide scientific information for the
potential use of a natural product as an alternative
treatment in improving testicular and sperm damages
caused by drug addiction
Methods
Animals
Male Sprague-Dawley rats aging 5 weeks and
weighing between 200-250 g from National Animal
Center, Salaya, Nakorn Pathom, Thailand were used
in this study The animals were housed at 24 ± 1 ◦C
under dark-light cycle 12:12 hours at Center for
Animal Research of Naresuan University All animals
were treated according to the guidelines for animal
care and use of laboratory animals, and the protocols
were approved by the Animals Research Committee
of Naresuan University, Thailand
Drug and reagent administrations
The drug and reagents used in this study were
described below;
hydrobromide was purchased from Sigma-
Aldrich® Lot#090M1298V
Univer-sity Hospital
purchased from Sigma Chemical Company, St
Louis, USA Amount of synthetic GABA was
equaled with the GABA found in PGBR GABA
was dissolved in distilled water before used
• Pre-germinated brown rice (PGBR) was supplied
by the Laboratory of Faculty of Agriculture
Natural Resources and Environment, Naresuan
University Briefly, Brown rice (Oryza sativa var
glutinosa) from KhekNoi, KhaoKho, Phetchabun
(Thailand) was soaked for 24 hours until
germ-inated PGBR was dried, ground to a powder
and suspended in distilled water before use
Experimental design
The animals were divided into 6 groups with 7
animals each and treated as described below (Figure
1);
• Control group (Control): Animals were treated
with normal saline by oral administration via
gavage for 75 days
treated with 30 mg/kg DXM via intraperitoneal
injection (i.p.) for 15 days and sacrificed on the
last day of treatment
days group (DW60): Animals were treated with
30 mg/kg DXM for 15 days (i.p.) and withdrawn for 60 days, respectively
(DD60): Animals were treated with 30 mg/kg DXM for 15 days (i.p.) and treated with 10 mg/
kg diazepam for 60 days by oral administration via gavage, respectively
group (DG60): Animals were treated with 30 mg/kg DXM for 15 days (i.p.) and treated with 0.8 mg/kg synthetic GABA for 60 days by oral administration via gavage, respectively
(DP60): Animals were treated with 30 mg/kg DXM for 15 days (i.p.) and treated with 5g/kg PGBR for 60 days by oral administration via gavage, respectively
Fig 1 The schematic diagram of experiment in rat model of addiction
Sample collection and Tissue preparation
After treatments, all animals were sacrificed by cervical dislocation The testes and epididymis were immediately dissected and weighed Sperm were released from cauda epididymis in phosphate- buffered saline (PBS) at 37 °C and assessed for the percentage of sperm motility The remaining samples were maintained and fixed in 10% formaldehyde for evaluating sperm morphology and concentration Each testis was cut, placed in cassettes and washed in PBS 3 times for 5 minutes Then, the tissues were processed for routine paraffin embedding until sectioning The tissue blocks were sectioned at 5 µm thickness and sections were floated on warm water (45 ◦C) in a water bath before mounting onto the microscope slides coated with 3-(Triethoxysilyl)- propylamine (S6225919 110; Merck, Hohenbrunn, Germany) The sections were allowed to dry overnight at room temperature before evaluating testicular structure and androgen receptor expression
Trang 3Sperm quality analysis
Sperm quality parameters including sperm
motility, sperm morphology and sperm concentration
were assessed The protocols for each parameter
followed the protocol described by Roboon et al
(2017) [10] briefly
Sperm motility
Spermatozoa were assessed for the percentage of
sperm motility using a Makler counting chamber and
counted under bright field (40X objective lens) The
motile and non-motile sperm were evaluated and
calculated
Sperm morphology
Spermatozoa were stained and assessed for the
percentage of normal sperm morphology with a total
count of 200 spermatozoa in each group
Sperm concentration
Sperm concentrations were assessed using a
hemocytometer and counting under bright field (40X)
The results of sperm concentration were reported in
term of epididymal sperm number (106cells)/ml
Testicular and epididymal structure analysis
Testicular and epididymal weight
Before being sacrificed, rats were weighed to
obtain body weight After that, the testes and
epididymes were immediately dissected and
weighed The data were evaluated and calculated as a
testicular/epididymal weight per body weight
Morphological changes of seminiferous tubules
Morphological changes of seminiferous tubules
were analyzed followed the protocols described by
Roboon et al (2017) [10] Briefly, two sections per
animal were used Each hematoxylin-eosin stained
section was evaluated under a light microscope
(Nikon eclipse 08i; Nikon, Bangkok, Thailand, Co.,
Ltd.) and a picture taken using a computerised image
capture system (Nikon digital camera DXM1200c,
Nikon, Bangkok, Thailand, Co., Ltd.) The data were
shown as percentage of each morphological type of in
seminiferous tubule per total number of seminiferous
tubules in each section
Immunohistochemistry analysis (Expression of
androgen receptor)
Androgen receptor (AR) expression was
detected in rat testis by using the indirect
immunohistochemistry technique Briefly, testicular
sections were deparaffinized and rehydrated After
that, the antigen was retrieved with high temperature
heating at 70P or 560 Watt in microwave for 5
minutes, 3 times Then, the sections were incubated with endogenous peroxidase blocking solution (10% methanol, 0.3% H2O2, 1% triton-X and PBS) for 30 minutes and washed with PBS 3 times for 5 minutes each The sections were incubated with non-specific protein blocking solution (5% normal goat serum and PBS) for 2 hours After that, the sections were incubated with AR primary antibody (Rabbit polyclonal IgG, Santa Cruz Biotechnology, USA and Merck Millipore, US) at a dilution of 1:25 in PBS containing 5% normal goat serum for 4 hours The sections were then washed with PBS 3 times and incubated with biotinylated secondary antibody for 2 hours and washed with PBS 3 times Then the sections were incubated with avidin-biotinylated horseradish peroxidase complex (ABC kit) (Vector Laboratories, Burlingame, California, USA) for 60 minutes and the sections were washed with PBS 3 times for 5 minutes each The specific proteins were visualized with chromogen 3,3’-Diaminobenzidine (DAB) (Vector Laboratories, Burlingame, California, USA) for 6 minutes and rinsed in distilled water for 5 minutes The sections were dehydrated in a series of increasing alcohol concentrations, and alcohol removed with xylene Finally, the tissues were mounted with mounting media (Fisher scientific, New Jersey, USA) Each immunostained section was evaluated under a light microscope at magnification 20X using the computerized image capture system Two sections per animal were used and 10 seminiferous tubules were randomly selected from each section AR immunopositive cells were analyzed by ImageJ software (http://rsb.info.nih.gov/ij/) The data were presented as percentage of positive cells per total cell number
Statistical analysis
Data were analyzed by one-way analysis of variance (ANOVA) The statistical significances were determined as p<0.05 The results were expressed as mean±SEM
Results
Sperm quality
Sperm motility Rat sperm motility in the DXM group was dramatically decreased (p<0.001) when compared to the control group Animal receiving DXM for 15 days and treated with diazepam and GABA standard for 60 days showed a recovery in the percentages of sperm motility but these were still significantly lower than the control Animals receiving DXM for 15 days and withdrawn from this drug for 60 days and animals treated with PGBR for 60 days showed percentages of
Trang 4recovery in sperm motility close to control values,
most notably in animals treated with PGBR (Table 1)
Sperm morphology
The percentage of normal sperm morphology in
animals treated with DXM alone also showed a highly
significant difference (p<0.001) when compared to the
control group The percentages of normal sperm
morphology in animals in all treated groups
gradually recovered when compared with animals
treated with DXM alone but still showed significant
differences when compared with the control
However, animals treated with PGBR showed the
most recovery in the percentage of normal sperm
morphology (Table 1)
Sperm concentration
Animal treated with DXM alone showed a
highly significant deficit in sperm concentration
(p<0.001) when compared with controls Animals in
the groups of DXM with drug withdrawal for 60 days,
and DXM with diazepam for 60 days also showed
significant differences from controls However,
animals treated with GABA standard and PGBR
showed no significant differences from the control
group (Table 1)
Testicular structure
Testicular and epididymal weight
Only in animals treated with DXM showed
significant changes in the testicular and epididymal
weight when compared with the control group There was a significant increase in the percentage of testicular weight/body weight (p<0.001), while the percentage of epididymal weight/body weight was decreased (Table 2)
Morphological changes of seminiferous tubules All parameters of morphological changes in seminiferous tubules showed significant increases in animals treated with DXM alone Additionally, vacuolization and irregular tubules were also signifi-cantly increased in animals treated with diazepam Apart from that, other treated groups showed a recovery effect of treatment reflected by no significant differences when compared with controls (Table 2)
Expression of androgen receptor (AR)
The percentages of AR expression in all cell types measured in animals receiving DXM alone was significant lower when compared to the control Similarly, significant decreases in AR expression in all cell types were also found in animals following drug withdrawal for 60 days and in animals treated with diazepam Animals treated with GABA standard and PGBR showed a recovery effect in some cell types; Leydig, Sertoli and peritubular myoid cells were not significantly changed when compared to controls, but spermatogonia, round spermatid and elongated spermatid were still significantly affected (Table 3)
Table 1 Sperm quality parameters measured in the control, Dextromethorphan only and treatment groups: Control,
Dextromethorphan only (DXM), Dextromethorphan+Drug withdrawal (DW60), Dextromethorphan+Diazepam (DD60), Dextromethorphan+GABA standard (DG60) and Dextromethorphan+Pre-germinated brown rice (DP60)
Sperm Motility (%) 65.07±3.37 2.86±2.86*** 56.94±0.01 48.35±2.52** 54.40±2.65* 60.74±2.51
Normal Sperm Morphology (%) 92.79±0.69 6.00±2.35*** 87.64±1.07*** 84.36±0.83*** 86.14±0.88*** 89.50±0.80*
Sperm Concentration
(x10 6 /ml) 260.06±8.05 18.19±3.01*** 215.68±7.87*** 215.74±5.70*** 252.30±3.84 254.54±3.05
Values are expressed as mean±SEM Statistical significance is indicated as *p<0.05, **p<0.01 and **p<0.001 in comparison with control group
Table 2 Testicular and epididymal weight and morphological changes of seminiferous tubules in the control, Dextromethorphan only and
treatment groups: Control, Dextromethorphan only (DXM), Dextromethorphan+Drug withdrawal (DW60), Dextromethorphan + Diazepam (DD60), Dextromethorphan + GABA standard (DG60) and Dextromethorphan+Pre-germinated brown rice (DP60)
Testicular weight/Body
weight 0.47±0.01 0.59±0.01*** 0.45±0.01 0.48±0.01 0.46±0.02 0.43±0.01
Epididymal
weight/Body weight 0.14±0.01 0.09±0.01*** 0.14±0.01 0.15±0.01 0.15±0.01 0.14±0.01
Separation of germinal
epithelium (%) 11.61±0.67 22.60±1.25*** 13.31±0.98 14.54±1.73 12.48±0.86 12.16±0.76
Vacuolization (%) 3.16±0.14 5.16±0.32*** 3.32±0.28 3.93±0.15** 3.19±0.09 3.07±0.11
Luminal sloughing of
germ cells (%) 0.51±0.04 1.31±0.18** 0.50±0.06 0.60±0.10 0.50±0.05 0.51±0.06
Irregular tubule
(%) 6.13±0.30 9.84±0.57*** 6.91±0.19 10.53±0.98*** 6.85±0.29 6.38±0.35
Atrophy (%) 0.10±0.05 0.29±0.07* 0.14±0.03 0.14±0.03 0.11±0.04 0.11±0.03
Values are expressed as mean±SEM Statistical significance is indicated as *p<0.05, **p<0.01 and **p<0.001 in comparison with control group
Trang 5Table 3 Percentages of androgen receptor (AR) expression in each cell type in the control, Dextromethorphan only and treatment
groups: Control, Dextromethorphan only (DXM), Dextromethorphan+Drug withdrawal (DW60), Dextromethorphan+Diazepam (DD60), Dextromethorphan+GABA standard (DG60) and Dextromethorphan+Pre-germinated brown rice (DP60)
Leydig cell 60.99±1.80 29.38±3.27*** 54.83±1.00* 44.09±1.92*** 58.24±0.70 58.43±1.21
Sertoli cell 58.96±0.94 32.09±2.46*** 58.77±0.27 43.61±1.38*** 59.42±0.40 58.37±0.52
Spermatogonia 15.91±1.09 2.61±0.51*** 7.01±0.41*** 5.07±0.29*** 10.17±0.44*** 13.38±0.25*
Round spermatid 17.45±0.25 8.07±0.685*** 12.72±0.15*** 13.28±0.46*** 14.44±0.42*** 15.03±0.27*** Elongated spermatid 57.24±0.67 40.08±0.32*** 53.83±0.95* 52.66±0.64** 54.27±0.99* 54.29±0.64*
Peritubular myoid cell 15.63±0.25 10.13±0.07*** 12.62±0.61** 11.41±0.51*** 14.76±0.90 15.07±0.49
Values are expressed as mean±SEM Statistical significance is indicated as *p<0.05, **p<0.01 and **p<0.001 in comparison with control group
Fig 2 Morphological changes of seminiferous tubules found in the control and all treatment groups; normal morphology of seminiferous tubule (a), separation of
germinal epithelium (arrow) (b), vacuolization of seminiferous tubule (arrow) (c), luminal sloughing of germ cells (arrow) (d), irregular tube of seminiferous tubule (circle) (e) and seminiferous tubule atrophy (circle) (f) (magnification 10X) at scale bar 25µm
Discussion
Effect of DXM on sperm quality, testicular
structure and androgen receptor expression
The present study demonstrated that DXM has
negative effects on sperm quality such as sperm
motility, sperm morphology and sperm concentration
all of which showed significant decreases when
compared with the control group The results were in
agreement with a previous study reporting decreases
of sperm motility and normal sperm morphology after DXM [11] This study reported that DXM can reduce gonadotropin-releasing hormone (GnRH) immunoreactivity in the hypothalamus [11] The reduction of GnRH may affect the function of anterior pituitary gland to secrete lutinizing hormone (LH) and follicle stimulating hormone (FSH) leading to dysfunction of testosterone production which regulates testicular development and testicular function [12] especially spermatogenesis Thus,
Trang 6reduced sperm quality may be an adverse
consequence of the pharmacological action of DXM
The percentage of testicular weight per body
weight (BW) after 30 mg/kg DXM for 15 days showed
a significant increase when compared with the control
group Similarly, morphological changes of
seminiferous tubules of these animals showed
significant increases in all parameters investigated
when compared with the control group These results
may reflect abnormal testicular development due to a
reduction of testosterone concentration [13] or the
reduction of hormonal receptors in the testis [14] or
the changes of sperm-related proteins which are
essential for sperm development [15] Furthermore,
the reduction of the peritubular myoid cell
contraction results in a congestion of the seminiferous
fluid that may cause vacuolization [16], this could
relate to the reduction of AR expression in peritubular
myoid cells found in the present study Although the
percentage of epididymal weight per body weight
was decreased in the DXM-treated group, this could
reflect the lack of content inside the epididymal
lumen due to a reduction in sperm production and
testicular secretions It has been suggested that
exposure to environmental toxicants increases
testic-ular oxidative stress, which leads to an increase in
germ cell apoptosis and subsequent
hyposperma-togenesis [17]
The expression of AR in testicular tissue of rats
induced by 30 mg/kg DXM for 15 days showed a
significant decrease of immunopositive cells in all cell
types when compared with the control group The reduction of immunopositive Leydig cells may be due
to the reduction of GnRH in the hypothalamus [11] which may also decrease AR expression in Leydig cells The reduction of AR expression in these cells therefore induces the decrease of androgen secretion which may affect AR expression in Sertoli cells and other target cells of androgen action The reduction of
AR in Sertoli cells has been reported to affect the development of spermatogonia and the spermatogenic phenotype [18] Additionally, AR function in Sertoli cells is to regulate spermatid adhesion to the seminiferous epithelium [18] Consequently, the reduction of AR in Sertoli cells may influence sperm development as well as spermiation
Recovery effect of drug withdrawal on sperm quality, testicular structure and androgen receptor expression in rat model of addiction
In the present study, the sperm motility of DW60 showed no statistically significant difference when compared with the control group while sperm morphology and sperm concentration showed signifi-cant decreases It has been reported that DXM is rapidly absorbed within 30 minutes in the gastrointestinal tract The duration of DXM is about 3-6 hours and half-life of about 2-4 hours [19], while the entire process of spermatogenesis takes approxi-mately 21 days in rat Therefore, sperm motility may recover after some period of withdrawal but sperm morphology and sperm concentration may
demonstrate enduring damage following the period of DXM treatment
In addition, DW60 showed a significant decrease of immunopositive cells in Leydig cells, spermatogonia, round spermatid, elongate spermatid and peritubular myoid cells when compared with the control group These results may reflect a reduction of testosterone concentration leading to abnormalities of sperm morphology and sperm concentration
Recovery effect of diazepam on sperm quality, testicular structure and
androgen receptor expression in rat model of addiction
All parameters of sperm quality in DD60 showed significant decreases when compared with control group These could reflect the fact that diazepam acts as an aneugen in spermatogenesis, which may cause daughter cell aneuploidy [20]
The morphological changes of semini-ferous tubules in DD60 showed significant
Fig 3 Seminiferous tubule stained with indirect immunohistochemistry for anti-androgen
receptor technique Androgen receptor positive cell are located in nuclei of Leydig cell ( ),
Sertoli cell ( ), spermatogonia ( ), round spermatid ( ), elongated spermatid (*), and
peritubular myoid cell ( ) (magnification 10X) at scale bar 25 µm
Trang 7increases in the percentages of vacuolization and
irregular tubule when compared with the control
group These morphological changes of seminiferous
tubules may relate to decreased testosterone
concentration caused by DXM [13] Reduction of
testosterone concentration affects degeneration of
Sertoli cell-Sertoli cell junctions that involve in
morphological changes of seminiferous tubules In
addition, reduction of testosterone concentration
induced the degeneration of the peritubular myoid
cell contraction resulting in congestion of the
seminiferous fluid may cause vacuolization [16], that
related with reduction of AR expression in
peritubular myoid cell It is possible that diazepam
has not only shown its recovery effect on the
morphological changes of seminiferous tubules
resulted from DXM but also may have an adverse
effect on structure of seminiferous tubule which may
lead to spermatogenesis deficiency
Recovery effect of GABA on sperm quality,
testicular structure and androgen receptor
expression in rat model of addiction
In terms of sperm quality, DG60 showed no
statistically significant difference from controls only
in sperm concentration This could reflect a recovery
effect of GABA on the number of sperm production
but not in sperm morphology and sperm motility A
previous study showed that GABA can modulate
GnRH secretion [21] which, as mentioned, has an
important role in regulation of FSH, LH and
testosterone levels, the essential hormones for
spermatogenesis Moreover, the expression of GABA
receptors on germ cells has been reported [22, 23, 24]
and which may be directly involved in the regulation
of sperm quality
The percentage of testicular weight per body
weight (BW) and the morphological changes of
seminiferous tubules in DG60 showed showed no
statistically significant differences when compared
with control group Jackson and Kuehl (2002) [21]
reported can GABA modulate GnRH secretion
Therefore, GABA treatment for 60 days shows its
recovery effect on testicular structure after DXM
administration These may due to the recovery of AR
expression in Sertoli, Leydig and peritubular myoid
cells which may be involved in testicular structure
development
Recovery effect of PGBR on sperm quality,
testicular structure and androgen receptor
expression in rat model of addiction
In the present study, all parameters of sperm
quality, the percentage of testicular weight per body
weight (BW) and the morphological changes of
seminiferous tubules measured in DP60 showed no significant differences when compared with the control group These could reflect a recovery effect of
PGBR treatment in rats administered DXM It has
been reported that PGBR has GABA in high amounts, which can help in the regulation of sperm quality [10,
22, 23, 24, 25, 26] In addition, PGBR contains
α-tocopherol, pyridoxine and thiamine Previous studies showed that antioxidants can enhance sperm quality, especially γ-oryzanol and α-tocopherol [27,
28, 29] γ-Oryzanol has been reported to improve the sperm morphology from lipid peroxidation [28] In addition, supplementation of γ-oryzanol can increase sperm concentration and sperm motility Further-more, α-tocopherol in combination with α-tocopherol effectively improved semen quality [30] This may well contribute to the fact that prolonged treatment with PGBR can recover sperm quality and testicular structure development in this animal model of drug addiction
The expression of AR in DP60 showed significant decreases only in spermatogenic cells This
could reflect sensitivity of these cells to DXM
However, prolonged treatment with PGBR may recover AR expression in these spermatogenic cells as they may take longer to recover than Sertoli, Leydig and peritubular myoid cells
Conclusions
The present study showed that PGBR treatment can enhance recovery of sperm quality, of morphological changes of seminiferous tubules and of
AR expression in DXM-treated animals These results suggested that PGBR, containing high amounts of GABA and antioxidants, may enhance the function of GnRH and related hormones in the regulation of testicular development, testicular function and spermatogenesis In addition, prolonged PGBR administration showed no adverse effect on sperm quality, morphological changes of seminiferous tubules and AR expression Therefore, PGBR may be a novel therapeutic strategy for the consequences of drug addiction in the future
Acknowledgements
We would like to send our sincere thanks to Assoc Prof Dr Sudarat Jiamyangyuen, Faculty of Agriculture, NU for providing us the PGBR, NU hospital for providing us with diazepam and Faculty
of Medical Science, NU for all laboratory facilities This study was financially supported by Agricultural Research Development Agency (ARDA) Thailand and
NU Research Fund
Trang 8Author contributions
S Thanoi and S Nudmamud-Thanoi performed
the experimental designs J Roboon, S Numamud-
Thanoi and S Thanoi performed the experiments S
Thanoi wrote the manuscript with all authors having
read and approved the final manuscript
Competing Interests
The authors have declared that no competing
interest exists
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